Method for regulating and controlling hematopoietic balance based on Jak2 / Stat3-Hif1a signal channel and application thereof

Abstract

The invention provides a method for regulating and controlling hematopoietic balance based on a Jak2 / Stat3-Hif1a signal channel and application of the method, and belongs to the field of biotechnology and medicine. According to the method, by regulating and controlling a Jak2 / Stat3-Hif1a signal channel, hematopoietic system unbalance caused by factors such as acute inflammation or acute hypoxia is intervened or reversed. Aiming at the problems that in the prior art, mechanism cognition is not clear, intervention targets are not clear, pathway relevance research is insufficient and the like, the molecular mechanism of acute inflammation affecting hematopoietic balance is deeply explored, and the internal relation of a Jak2 / Stat3 pathway and Hif1alpha for regulating and controlling hematopoietic balance under the acute stress condition is disclosed. The invention can be applied to preparation of medicines for treating blood system diseases caused by factors such as acute inflammation and hypoxia, particularly provides a targeted intervention strategy for accurately correcting hematopoietic lineage migration aiming at diseases such as sepsis-related anemia, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medicine, and specifically relates to a method for intervening or reversing the imbalance of hematopoietic system caused by acute inflammation or acute hypoxia and the like by regulating Jak2 / Stat3-Hif1a signaling pathway, and application of the method in preparation of a medicament for treating related blood system diseases. BACKGROUND

[0002] Hematopoietic differentiation is a key physiological process throughout the life cycle of all vertebrates. The formation of blood cells is a dynamically regulated system involving self-renewal and lineage-specific differentiation of hematopoietic stem cells, ultimately generating various mature blood cells such as red blood cells, granulocytes and macrophages. Under steady-state conditions, the body maintains a balanced production of red blood cells and myeloid cells to ensure normal physiological function. Precise regulation of this red cell-myeloid hematopoietic balance is crucial for maintaining the health of the body.

[0003] Under pathological conditions, this delicate balance is easily disrupted. For example, in the acute inflammatory state induced by sepsis, acute infection and the like, the body's hematopoietic system undergoes significant restructuring, i.e., "hematopoietic lineage shift". Lipopolysaccharide is the main component of the outer membrane of gram-negative bacteria and is a potent inducer of inflammation, often used to construct an acute inflammatory model. Studies have shown that LPS-induced acute inflammation activates immune cells, leading to the release of a large number of pro-inflammatory cytokines, which can act on the hematopoietic microenvironment to inhibit the production of red blood cells, while possibly over-activating or abnormally activating the production of myeloid cells. Although the association between inflammation and hematopoietic disorder has been recognized, the underlying core molecular mechanisms and key signaling pathways have not been fully elucidated, which poses a challenge to the development of effective clinical intervention means.

[0004] In the complex network of hematopoietic regulation, Janus kinase 2 / signal transducer and activator of transcription 3 (JAK2 / STAT3) pathway and hypoxia-inducible factor 1 alpha (HIF1a) are two key nodes that have attracted much attention. The JAK2 / STAT3 pathway is a core signaling cascade downstream of various cytokines, which profoundly affects cell proliferation, differentiation and survival by regulating the expression of target genes such as bcl2, cMyc, etc. HIF1a is a core transcription factor for cells to respond to hypoxic environment, which can regulate a series of genes including erythropoietin, glycometabolism enzymes, angiogenic factors to adapt to low oxygen conditions. Previous studies have focused on the independent functions of these two pathways, for example, the role of JAK2 / STAT3 in inflammation signaling and the role of HIF1a in promoting red blood cell production under chronic hypoxia.

[0005] The prior art has the following deficiencies:

[0006] There is a lack of systematic research and clear evidence on how acute inflammation precisely breaks the erythroid-myeloid hematopoietic balance, especially the functional cross or synergy of JAK2 / STAT3 and HIF1a pathways in the process. Due to the lack of understanding of the core regulatory mechanism, the current treatment strategies for inflammation-related blood diseases are mostly supportive or non-specific, lacking targeted drugs that can precisely correct the hematopoietic lineage shift.

[0007] The prior art fails to fully reveal the internal relationship between the JAK2 / STAT3 pathway and HIF1a in regulating hematopoietic balance under acute stress conditions, especially in non-chronic hypoxia scenarios, the mechanism of how HIF1a is activated and affects hematopoietic differentiation is not clear. SUMMARY

[0008] The present application provides a method for regulating hematopoietic balance based on Jak2 / Stat3-Hif1a signaling pathway and its application. The present application found that LPS inflammation and hypoxia conditions can affect hematopoietic balance through Jak2 / Stat3-Hif1a signaling axis, leading to decreased expression of red blood cells and increased expression of neutrophils and macrophages, thereby breaking the erythroid-myeloid hematopoietic balance, including the following steps:

[0009] Wild-type zebrafish and three transgenic zebrafish lines Tg(gata1a:DsRed), Tg(lyz:EGFP) and Tg(mpeg1:EGFP) were used to observe the expression of red blood cells, neutrophils and macrophages.

[0010] Whole mount in situ hybridization (WISH) technique was used to detect the expression position and abundance of hif1a gene in zebrafish embryos. O-dianisidine staining, Sudan black staining, and neutral red staining were used to detect the expression of red blood cells, neutrophils and macrophages in zebrafish embryos.

[0011] Microinjection technology was used to inject LPS or sihif1a into 3dpf large zebrafish larvae to establish an inflammation model in zebrafish and knock down the hif1a gene.

[0012] K562 cells and THP-1 cells were treated with LPS, and Western blot technique was used to detect the expression of HIF1a and JAK2 / STAT3 proteins. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is shown that LPS inflammation inhibits the generation of red blood cells in zebrafish.

[0014] Figure 2LPS inflammation was shown to promote neutrophil production in zebrafish.

[0015] Figure 3 LPS was shown to promote macrophage development in zebrafish.

[0016] Figure 4 Differential gene expression analysis was shown.

[0017] Figure 5 Clustered heat map analysis was shown.

[0018] Figure 6 LPS was shown to promote expression of HIF1a.

[0019] Figure 7 LPS inflammation was shown to positively regulate expression of HIF1a through the JAK-STAT pathway.

[0020] Figure 8 LPS inflammation was shown to positively regulate expression of HIF1a through the JAK-STAT pathway at the zebrafish level.

[0021] Figure 9 Inhibition of the HIF1a gene and the JAK-STAT pathway was shown to reverse the decrease in red blood cell production in zebrafish embryos caused by LPS inflammation.

[0022] Figure 10 Inhibition of the HIF1a gene and the JAK-STAT pathway was shown to reverse the increase in neutrophil production in zebrafish embryos caused by LPS inflammation.

[0023] Figure 11 Inhibition of the HIF1a gene and the JAK-STAT pathway was shown to reverse the increase in macrophage production in zebrafish embryos caused by LPS inflammation.

[0024] Figure 12 Hypoxia was shown to positively regulate expression of HIF1a through the JAK-STAT pathway.

[0025] Figure 13 Hypoxia was shown to positively regulate expression of HIF1a through the JAK-STAT pathway at the zebrafish level.

[0026] Figure 14 Inhibition of the HIF1a gene and the JAK-STAT pathway was shown to reverse the decrease in red blood cell production in zebrafish embryos caused by hypoxia.

[0027] Figure 15 Inhibition of the HIF1a gene and the JAK-STAT pathway was shown to reverse the increase in neutrophil production in zebrafish embryos caused by hypoxia.

[0028] Figure 16It is demonstrated that inhibition of HIF1a gene and JAK-STAT pathway can reverse the increase of macrophages in zebrafish embryos caused by hypoxia. DETAILED DESCRIPTION

[0029] Example 1

[0030] Wild type zebrafish were randomly divided into normal group and LPS group. The erythrocytes in zebrafish embryos were specifically stained and quantitatively analyzed by o-dianisidine staining method to evaluate the changes of erythropoiesis. The staining results showed that LPS significantly inhibited the number of erythrocytes in the caudal hematopoietic tissue of zebrafish embryos Figure 1 A). At the same time, we also used Tg(gata1a:DsRed) transgenic zebrafish which specifically labeled erythrocytes to observe the effect of LPS on the development of erythrocytes by detecting the changes of fluorescence expression intensity. By analyzing the fluorescence intensity of the normal group and the LPS group, we found that the red fluorescence intensity of the LPS group was significantly lower than that of the normal group Figure 1 B). The qPCR detection results also showed that the expression of zebrafish erythroid transcription factor gata1a and hemoglobin hbae3 decreased in the LPS group Figure 1 C). The o-dianisidine staining results, fluorescence expression analysis of transgenic lines and qPCR detection results were consistent, which indicated that the inflammatory response induced by LPS significantly inhibited the generation of erythrocytes in zebrafish embryos.

[0031] Example 2

[0032] Using wild type zebrafish model, neutral granulocytes were quantitatively analyzed by Sudan black staining method. The results showed that the number of neutral granulocytes in the yolk sac of the LPS injection group was significantly increased Figure 2 A). Then we used Tg(lyz:EGFP) transgenic zebrafish which labeled neutral granulocytes to quantitatively analyze the changes of the number of neutral granulocytes by detecting the expression intensity of green fluorescent protein. The fluorescence intensity analysis results were consistent with the Sudan black staining results, both of which showed that the number of neutral granulocytes in the yolk sac of the LPS injection group was significantly higher than that of the normal group Figure 2 B). The qPCR detection results also showed that the expression of zebrafish granulocyte colony-stimulating factor csf3a and myeloperoxidase mpx increased in the LPS group Figure 2 C). These results indicated that LPS treatment significantly increased the number of neutral granulocytes in zebrafish embryos.

[0033] Example 3

[0034] Wild type zebrafish were divided into normal group and LPS injection group, and the number of macrophages in zebrafish embryos was shown by neutral red staining. The staining results showed that the number of macrophages in the hindbrain ventricle of zebrafish embryos in the LPS group was significantly increased Figure 3A). Similarly, we also used the transgenic zebrafish line Tg(mpeg1:EGFP) to observe the development of macrophages. The green fluorescence intensity analysis results showed that the fluorescence intensity in the injection site of LPS group zebrafish was significantly higher than that of the normal group( Figure 3 B). The qPCR detection results also showed that the expression of nos2b in the macrophages of LPS group zebrafish increased. Figure 3 C). These results showed that LPS treatment could lead to an increase in the number of macrophages in zebrafish embryos.

[0035] Example 4

[0036] The LPS-related data sets were collected using the NCBIGEO database, and the GSE60290 data set for microarray chip data analysis of mouse bone marrow-derived macrophages and the GSE19315 data set for microarray chip data analysis of THP-1 cells were obtained. At the same time, the GEO2R of the GEO platform was used to analyze the differential gene expression of the two data sets, and the Sangerbox 3.0 online platform was used for visualization of differential genes. The differential gene analysis results showed that the expression of HIF1a, JAK2 and STAT3 genes in the LPS group of GSE6029 data set and GSE19315 data set was higher than that in the control group( Figure 4 ).

[0037] Example 5

[0038] From the differentially expressed genes, 20 up-regulated and 20 down-regulated genes were selected, and the expression of 40 genes was obtained using the expression data of each group of genes in the data set. The data was imported into the Euichun platform for cluster heat map analysis, and the differential genes in the two data sets were visualized( Figure 5 ).

[0039] Example 6

[0040] K562 cells and THP-1 cells were used as cell models, and the protein expression level of HIF1a was detected by Western blot experiment. In K562 cells, we used a working concentration of 10 μg / ml LPS solution to treat for 6 h to establish an inflammatory cell model; in THP-1 cells, we used 100 ng / ml PMA to induce differentiation into macrophages for 24 h, and then used 1 μg / ml LPS solution to treat for 24 h to establish an inflammatory model. Western blot results showed that whether in K562 cells( Figure 6 A), or in THP-1 cells( Figure 6B), the expression of HIF1a in both cells showed a trend of increase after LPS induction. This result indicates that LPS inflammation can affect hematopoiesis by stabilizing the expression of HIF1a.

[0041] Example 7

[0042] Ruxolitinib was used to inhibit the JAK-STAT pathway, and the relationship between HIF1a and the JAK-STAT pathway was studied by Western blot. Ruxolitinib is a selective kinase inhibitor that inhibits Janus-associated kinases (JAKs) JAK1 and JAK2, and plays a role by blocking the JAK signal transduction and transcription activator (STAT) pathway. In K562 cells and THP-1 cells, 10 μM ruxolitinib was used to inhibit LPS inflammation, and the expression levels of HIF1a, p-STAT3, STAT3, p-JAK2, and JAK2 proteins were detected. Western blot results show that LPS treatment of both cells can cause an increase in the expression of HIF1a, p-STAT, and p-JAK2, while treatment with ruxolitinib can reverse the increase in the expression of both caused by LPS Figure 7 ). This result shows that inhibition of the JAK-STAT pathway also inhibits the expression of HIF1a, i.e. LPS inflammation can positively regulate the expression of HIF1a through the JAK-STAT pathway.

[0043] Example 8

[0044] Zebrafish, a model organism, was used to study hematopoietic differentiation. By microinjection of sihif, hif1a gene silencing was achieved at the zebrafish level; zebrafish were soaked in 100 μM concentration of ruxolitinib to inhibit the JAK-STAT pathway. The expression of HIF1a was stabilized by LPS inflammation through the JAK-STAT pathway at the zebrafish level. After 6 hours of treatment with normal zebrafish, LPS, LPS+sihif, and LPS+ruxolitinib groups, in situ hybridization was used to show the expression position and abundance of hif1a gene on zebrafish. In situ hybridization results show that compared with the normal group, LPS treatment can increase the expression of hif1a gene Figure 8 A and B). Compared with the LPS treatment group, the expression of hif1a gene in the LPS+sihif group decreased, achieving hif1a gene silencing at the zebrafish level; the expression of hif1a gene in the LPS+ruxolitinib group also decreased Figure 8 A and B).

[0045] Example 9

[0046] The wild type zebrafish was used for o-anisidine staining to show the number of red blood cells, combined with transgenic line Tg(gata1a:DsRed) to study the effect of HIF1a gene and JAK-STAT pathway on erythropoiesis under LPS-induced inflammatory conditions. Compared with the LPS treatment group, the staining area of the caudal hematopoietic tissue in the sihif injection group and the ruxolitinib soaking group was significantly increased, and the red fluorescence intensity in the transgenic zebrafish was significantly increased (A and B). This result shows that inhibiting HIF1a gene and JAK-STAT pathway can reverse the reduction of red blood cell production in zebrafish embryos caused by LPS inflammation. Figure 9 A and B). This result shows that inhibiting HIF1a gene and JAK-STAT pathway can reverse the reduction of red blood cell production in zebrafish embryos caused by LPS inflammation.

[0047] Example 10

[0048] The generation of neutrophils was studied by zebrafish Sudan black staining and transgenic line Tg(lyz:EGFP). Sudan black staining results show that compared with the LPS group, the number of neutrophils in the yolk sac of the sihif injection group and the ruxolitinib soaking group was significantly reduced (A). The fluorescence intensity in the yolk sac of the transgenic zebrafish embryos was also significantly reduced (B). These results show that inhibiting hif gene and JAK-STAT pathway can reverse the increase in neutrophils caused by LPS inflammation. Figure 10 A). The fluorescence results of the transgenic zebrafish show that the fluorescence intensity in the yolk sac of the sihif group and the ruxolitinib soaking group was significantly reduced compared with the LPS group (B). Consistent with the results of neutrophils, silencing hif gene and inhibiting JAK-STAT pathway can reverse the increase in macrophages caused by LPS inflammation. Figure 10 B). These results show that inhibiting hif gene and JAK-STAT pathway can reverse the increase in neutrophils caused by LPS inflammation.

[0049] Example 11

[0050] The generation of macrophages was shown by neutral red staining and transgenic zebrafish Tg(mpeg1:EGFP). The staining results show that compared with the LPS group, the number of macrophages in the hindbrain ventricle of the sihif group and the ruxolitinib soaking group was significantly reduced (A). The fluorescence results of the transgenic zebrafish show that the fluorescence intensity in the yolk sac of the sihif group and the ruxolitinib soaking group was significantly reduced compared with the LPS group (B). Consistent with the results of neutrophils, silencing hif gene and inhibiting JAK-STAT pathway can reverse the increase in macrophages caused by LPS inflammation. Figure 11 A). The fluorescence results of the transgenic zebrafish show that the fluorescence intensity in the yolk sac of the sihif group and the ruxolitinib soaking group was significantly reduced compared with the LPS group (B). Consistent with the results of neutrophils, silencing hif gene and inhibiting JAK-STAT pathway can reverse the increase in macrophages caused by LPS inflammation. Figure 11 B). These results show that inhibiting hif gene and JAK-STAT pathway can reverse the increase in macrophages caused by LPS inflammation.

[0051] Example 12

[0052] In K562 cells and THP-1 cells, we used a 1% O2 concentration of the hypoxic incubator to culture cells for 24 h, used 10 μM of ruxolitinib to inhibit the JAK-STAT pathway, and detected the expression levels of HIF1α, p-STAT3, STAT3, p-JAK2, and JAK2 proteins. Western blot results showed that hypoxic treatment of the two cells can cause the expression of HIF1α, p-STAT, and p-JAK2 to increase, and the treatment of ruxolitinib can reverse the increase in the expression of both caused by hypoxia Figure 12 ). This result shows that the inhibition of the JAK-STAT pathway also inhibits the expression of HIF1α, i.e., hypoxia can positively regulate the expression of HIF1α through the JAK-STAT pathway.

[0053] Example 13

[0054] The zebrafish was placed in a 5% O2 concentration of the hypoxic incubator for 12 h to establish an acute hypoxia model, the hif gene silencing in zebrafish was achieved by microinjection of sihif, and the JAK-STAT pathway was inhibited by soaking in 100 μM of ruxolitinib. The expression abundance and location of the hif1a gene in the normal group, the hypoxia group, the hypoxia + sihif group, and the hypoxia + ruxolitinib group were shown by in situ hybridization. Compared with the normal group, the expression of the hif1a gene in the hypoxia group increased significantly Figure 13 A and B); compared with the hypoxia group, the expression of the hif1a gene in the sihif injection group and the ruxolitinib soaking group showed a downward trend Figure 13 A and B).

[0055] Example 14

[0056] The number of red blood cells in the caudal hematopoietic tissue of zebrafish in each group was shown by o-dianisidine staining; the double-fluorescent zebrafish labeled with red blood cells and neutrophils was obtained by mating two transgenic lines of Tg(gata1a:DsRed) and Tg(lyz:EGFP) zebrafish, and the red fluorescence intensity in the caudal hematopoietic tissue of the double-fluorescent zebrafish reflected the generation of red blood cells, and the green fluorescence intensity reflected the generation of neutrophils. The staining results showed that the number of red blood cells in the zebrafish after hypoxic treatment decreased significantly, and the injection of sihif and the soaking of ruxolitinib can reverse the decrease in the number of red blood cells in zebrafish caused by hypoxia Figure 14 A). The analysis results of red fluorescence intensity showed that the fluorescence intensity of the zebrafish in the hypoxia group decreased significantly, and the red fluorescence intensity of the zebrafish in the sihif injection group and the ruxolitinib soaking group increased compared with the hypoxia group Figure 14 B). These results show that hypoxic treatment can also stabilize the expression of HIF1α through the JAK-STAT pathway to cause a decrease in the generation of red blood cells.

[0057] Example 15

[0058] The number of neutrophils in the caudal hematopoietic tissue of zebrafish embryos was shown by Sudan black staining, and the zebrafish of two transgenic lines Tg(gata1a:DsRed) and Tg(lyz:EGFP) were crossed to obtain double-fluorescent zebrafish labeling red blood cells and neutrophils, and the green fluorescently labeled neutrophils. The results of Sudan black staining showed that hypoxia treatment increased the number of neutrophils in the caudal hematopoietic tissue of zebrafish embryos, and injection of sihif and immersion of Ruxolitinib could reverse the increase of neutrophils caused by hypoxia Figure 15 ).

[0059] Example 16

[0060] The number of macrophages in the zebrafish lateral ventricle was shown by neutral red staining, and the transgenic zebrafish Tg(mpeg1:EGFP) labeling macrophages was used to show the generation of macrophages in the caudal hematopoietic tissue. Both the staining and fluorescence intensity analysis results showed that hypoxia treatment significantly increased the number of macrophages, and after injection of sihif and immersion of Ruxolitinib, the increase of macrophages caused by hypoxia showed a downward trend Figure 16 A and B).

[0061] The sequence of zebrafish WISH probe primers is shown in Table 1 Table 1 Zebrafish WISH probe primers

[0062] The sequence of zebrafish gene amplification primers is shown in Table 2

Claims

1. A method for regulating hematopoietic balance based on Jak2 / Stat3-Hif1a signal pathway, characterized in that, By regulating Jak2 / Stat3-Hif1a signal pathway, the hematopoietic system imbalance caused by acute inflammation or acute hypoxia and other factors is intervened or reversed.

2. The method of claim 1, wherein, The acute inflammation is manifested by a significant increase in the number of white blood cells, and the acute hypoxia is manifested by excessive proliferation of red blood cells.

3. The method of claim 1, wherein, The regulation includes inhibiting or activating key molecules in the Jak2 / Stat3-Hif1a signal pathway to correct the hematopoietic lineage shift.

4. The method of claim 1, wherein, The hematopoietic system imbalance includes sepsis-related anemia, renal anemia or other blood system diseases caused by acute inflammation and hypoxia.

5. The use of a method for regulating hematopoietic balance based on the Jak2 / Stat3-Hif1a signal pathway in the preparation of a drug for treating hematological diseases, characterized in that, The drug intervenes or reverses the hematopoietic system imbalance caused by acute inflammation or acute hypoxia and other factors by regulating the Jak2 / Stat3-Hif1a signal pathway.

6. Use according to claim 5, characterized in that, The blood system disease includes sepsis-related anemia, renal anemia or other blood system diseases caused by acute inflammation and hypoxia.

7. Use according to claim 5, characterized in that, The drug is a small molecule compound, a biological preparation or an active ingredient of traditional Chinese medicine that targets the Jak2 / Stat3-Hif1a signal pathway.

8. Use according to claim 5, characterized in that, The drug restores the normal differentiation function of hematopoietic stem cells by improving the hematopoietic microenvironment under acute inflammation or acute hypoxia conditions.

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